Fanconi anemia (FA) is a recessively inherited disease characterized by congenital defects, bone marrow failure, and cancer susceptibility. Thirteen genes have now been described that are mutated to cause FA, and three are discovered by my group. Recent evidence suggests that FA proteins function in a DNA damage response pathway involving the proteins produced by the breast cancer susceptibility genes BRCA1 and BRCA2. A key step in that pathway is a modification of an FA protein, FANCD2. The modification, monoubiquitylation, results in redistribution of FANCD2 to specific spots in the nucleus where BRACA1 also localizes. Five other FA proteins (FANCA, -C, -E, -F, and -G) have been found to interact with each other to form a multiprotein nuclear complex, the FA core complex. This complex functions upstream in the pathway and is required for FANCD2 monoubiquitylation. We have purified the FA protein core complex and found that it contains four new components in addition to the five known FA proteins. One new component of this complex, termed PHF9, possesses ubiquitin ligase activity in vitro and is essential for FANCD2 monoubiquitylation in vivo. PHF9 is defective in a cell line derived from a Fanconi anemia patient, and therefore represents a novel Fanconi anemia gene (FANCL). Our data suggest that PHF9 plays a crucial role in the Fanconi anemia pathway as the catalytic subunit required for FANCD2 monoubiquitylation. The discovery of PHF9/FANCL might provide a potential target for new therapeutic modalities. We then showed that the 95 kd subunit of the Fanconi anemia core complex is defective in FA complementation group B patients (the gene is named FANCB). FANCB is X-linked and present in only one active copy. Thus, FANCB could represent a vulnerable target in the machinery that maintains genome stability, because it will only take one mutation to inactivate FANCB, compared to two mutations required to inactivate other FA genes. We demonstrated that the 250 Kda subunit of the FA core complex, FAAP250, is mutated in FA patients of a new complementation group, FA-M. The gene encoding the FAAP250 protein was renamed FANCM. FANCM has a conserved helicase domain and a DNA-translocase activity. FANCM has at least three important roles in the FA DNA damage response pathway. First, FANCM plays a structural role to allow assembly of the FA core complex, because in its absence, the nuclear localization and stability of several FA proteins are defective. Second, FANCM translocates and remodels various DNA structures, which may be important for subsequent DNA repair. Third, FANCM is hyperphosphorylated in response to DNA damage, suggesting that it may serve as a signal transducer through which the activity of the core complex is regulated. We have identified the 100 Kda subunit of the FA core complex, FAAP100, and shown that this protein is required for stability and a key function of the complex--FANCD2 monoubiquitination. We recently have identified the 24 Kda subunit of the FA core complex, termed FAAP24. FAAP24 contains an ERCC4-like endonuclease domain, and forms a heterodimer with FANCM. We find that FAAP24 can recognize structured DNA that mimics intermediates generated during DNA replication. Moreover, it can target FANCM to such structures. Cells depleted of FAAP24 show phenotypes that are characteristics of FA cells. Our results demonstrate that FAAP24 is a new essential component of the FA core complex, and its defect could cause FA. We also collaborated with other labs to demonstrate that PALB2, a partner of BRCA2, is the gene defective in Fanconi anemia complementation group N patients. We demonstrated that FANCM possesses an ATP-independent binding activity and an ATP-dependent bi-directional branch-point translocation activity on a synthetic four-way junction DNA, which mimics intermediates generated during homologous recombination or at stalled replication forks. Using an siRNA-based complementation system, we found that the ATP-dependent activities of FANCM are required for cellular resistance to a DNA crosslinking drug, mitomycin C (MMC), but not for the monoubiquitination of FANCD2 and FANCI. In contrast, monoubiquitination requires the entire helicase domain of FANCM, which has both ATP- dependent and independent activities. These data are consistent with participation of FANCM and its associated FA core complex in the FA pathway at both signaling through monoubiquitination and the ensuing DNA repair. We identified two new components in the FA core complex, MHF1 and MHF2. These two proteins form a histone-fold heterodimer and associates with FANCM to form a conserved DNA-remodeling complex. We foud that MHF stimulates DNA binding and replication fork remodeling by FANCM. In the cell, FANCM and MHF are rapidly recruited to forks stalled by DNA interstrand crosslinks, and both are required for cellular resistance to such lesions. In vertebrates, FANCM-MHF associates with the Fanconi anemia (FA) core complex, promotes FANCD2 monoubiquitination in response to DNA damage, and suppresses sister-chromatid exchanges. Yeast orthologs of these proteins function together to resist MMS-induced DNA damage and promote gene conversion at blocked replication forks. Thus, FANCM-MHF is an essential DNA-remodeling complex that protects replication forks from yeast to human. We found that another FA protein, FANCJ, becomes hyperphosphorylated in response to DNA damage. We are investigating if this phosphorylation regulates activity of FANCJ in DNA repair. We collaborated with Dr. Lei Li's lab to develop a chromatin-IP-based strategy termed eChIP and detected association of multiple FA proteins with DNA crosslinks in vivo. We used this new method to investigate the mechanism of how various FA proteins are recruited to the interstrand DNA crosslinks that block replication and transcription. Interdependence analyses revealed that crosslink-specific enrichment of various FA proteins is controlled by distinct mechanisms. BRCA-related FA proteins (BRCA2, FANCJ/BACH1, and FANCN/PALB2), but not FA core and I/D2 complexes, require replication for their crosslink association. FANCD2, but not FANCJ and FANCN, requires the FA core complex for its recruitment. FA core complex requires nucleotide excision repair proteins XPA and XPC for its association. Consistent with the distinct recruitment mechanism, recombination-independent crosslink repair was inversely affected in cells deficient of FANC-core versus BRCA-related FA proteins. Thus, FA proteins participate in distinct DNA damage response mechanisms governed by DNA replication status. We are continuing to identify and characterize additional components of the FA complex. The eventual goal is to find new targets for drug interventions.